library(magrittr)
library(tidyverse)
library(Seurat)
library(readxl)
library(cowplot)
library(colorblindr)
library(viridis)
library(grid)
library(ggpubr)

pcut <- params$pcut
## load global vars: 
source("_src/global_vars.R")

# meta_tbl
# clrs
# markers_v6
# markers_v6_super
# cell_type_super_lookup
## load data
seu_obj_cc <- read_rds("/work/shah/uhlitzf/data/SPECTRUM/freeze/v6/Ovarian.cancer.cell_processed_filtered.rds")

seu_obj_ml <- read_rds("/work/shah/uhlitzf/data/SPECTRUM/freeze/v6/Myeloid.super_processed_filtered_annotated.rds")

seu_obj_fb <- read_rds("/work/shah/uhlitzf/data/SPECTRUM/freeze/v6/Fibroblast.super_processed_filtered_annotated.rds")

gois <- c(grep("^ALDH", rownames(seu_obj_cc), value = T),
          "PROM1", "CD68", "PECAM1", "NT5E") %>% .[!str_detect(. ,"-AS")]

myfeatures <- c("sample", "umapharmony_1", "umapharmony_2", "cell_type", "cluster_label")

get_data_wrapper <- . %>%
  FetchData(c(myfeatures, gois)) %>%
  as_tibble() %>%
  gather(gene, value, -c(1:length(myfeatures))) %>%
  left_join(meta_tbl, by = "sample")

plot_data <- bind_rows(
  `Ovarian.cancer.cell` = get_data_wrapper(seu_obj_cc),
  `Fibroblast` = get_data_wrapper(seu_obj_fb),
  `Myeloid.cell` = get_data_wrapper(seu_obj_ml),
  .id = "cell_type_super"
)

plot_data_full <- plot_data
plot_data_sub <- sample_n(plot_data, 100000)
plot_data <- plot_data_sub
write_tsv(plot_data_sub, "cache.tsv")

plot_data <- read_tsv("cache.tsv")
plot_data <- plot_data_full

1 Overview

1.1 considered data

We assess marker gene expression in a HGSOC cohort of 42 patients across major cell types:

  • Fibroblasts: 154k
  • Myeloid cells: 162k
  • Cancer cells: 214k

Marker genes:

cat(gois, sep = ", ")
## ALDH4A1, ALDH9A1, ALDH1L1, ALDH7A1, ALDH5A1, ALDH8A1, ALDH1B1, ALDH1A1, ALDH3B2, ALDH3B1, ALDH18A1, ALDH1L2, ALDH2, ALDH6A1, ALDH1A2, ALDH1A3, ALDH3A2, ALDH3A1, ALDH16A1, PROM1, CD68, PECAM1, NT5E

1.2 Marker percentage

plot_data_comp <- plot_data %>% 
  mutate(expressed = value > 0) %>% 
  group_by(patient_id_short, cell_type_super, gene) %>% 
  summarise(pct.expressed = sum(expressed)/length(expressed))

write_tsv(plot_data_comp, "mehta_marker_expression_pct.tsv")

ggplot(plot_data_comp) + 
  geom_boxplot(aes(gene, pct.expressed)) + 
  facet_grid(~cell_type_super) +
  theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0))

2 Detailed single cell marker expression data

cluster_label_clrs <- c(clrs$cluster_label$Fibroblast.super,
                        clrs$cluster_label$Myeloid.super,
                        clrs$cluster_label$Ovarian.cancer.super)

plot_data <- plot_data %>% 
  mutate(cluster_label = ordered(cluster_label, levels = names(cluster_label_clrs)))

common_layers <- list(
  remove_xaxis,
  remove_yaxis
)

ggplot(distinct(plot_data, umapharmony_1, umapharmony_2, cell_type_super, .keep_all = T)) + 
  geom_point(aes(umapharmony_1, umapharmony_2, 
                 color = cluster_label), size = 0.01, alpha = 0.2) +
  facet_grid(~cell_type_super) +
  scale_color_manual(values = cluster_label_clrs) + 
  guides(color = guide_legend(override.aes = list(size = 3, alpha = 1))) +
  common_layers

2.1 in the embedding

ggplot() + 
  geom_point(aes(umapharmony_1, umapharmony_2), 
             size = 0.01, alpha = 0.2, color = "grey80",
             data = filter(plot_data, value == 0)) +
  geom_point(aes(umapharmony_1, umapharmony_2, 
                 color = value), size = 0.01, alpha = 0.2, 
             data = filter(plot_data, value > 0)) +
  facet_grid(gene~cell_type_super) +
  scale_color_viridis() + 
  common_layers

2.2 box plot distributions wrt cell type subclusters

ggplot(plot_data) + 
  geom_boxplot(aes(cluster_label, value, fill = cluster_label)) + 
  facet_grid(gene~cell_type_super, space = "free", scales = "free") +
  theme(axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0)) +
  guides(fill = F) +
  scale_fill_manual(values = cluster_label_clrs)

3 session info

devtools::session_info()
## ─ Session info ───────────────────────────────────────────────────────────────
##  setting  value                       
##  version  R version 3.6.2 (2019-12-12)
##  os       Debian GNU/Linux 10 (buster)
##  system   x86_64, linux-gnu           
##  ui       X11                         
##  language (EN)                        
##  collate  en_US.UTF-8                 
##  ctype    en_US.UTF-8                 
##  tz       Etc/UTC                     
##  date     2021-02-22                  
## 
## ─ Packages ───────────────────────────────────────────────────────────────────
##  package      * version    date       lib
##  abind          1.4-5      2016-07-21 [2]
##  ape            5.3        2019-03-17 [2]
##  assertthat     0.2.1      2019-03-21 [2]
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##  bibtex         0.4.2.2    2020-01-02 [2]
##  Biobase        2.46.0     2019-10-29 [2]
##  BiocGenerics   0.32.0     2019-10-29 [2]
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## [1] /home/uhlitzf/R/lib
## [2] /usr/local/lib/R/site-library
## [3] /usr/local/lib/R/library